Causative microorganisms of hospital-acquired pneumonia and laboratory procedures for isolation and identification of causative microorganisms

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1 Blackwell Science, LtdOxford, UKRESRespirology Blackwell Science Asia Pty LtdMarch 20049S1S6S12Original Article S Kohno et al. Respirology (2004) 9, S6 S12 CHAPTER 3 and laboratory procedures for isolation and identification of causative microorganisms The committee for The Japanese Respiratory Society guidelines in management of respiratory infections The Japanese Respiratory Society and laboratory procedures for isolation and identification of causative microorganisms The committee for The Japanese Respiratory Society guidelines in management of respiratory infections. Respirology 2004; 9: S6 S12 APPROACHES TO ISOLATION AND IDENTIFICATION OF CAUSATIVE MICROORGANISMS Special pathogenic microorganisms, such as opportunistic pathogens, Mycobacterium and fungi are more frequent causes of hospital-acquired pneumonia (HAP) than of community-acquired pneumonia (CAP), and because of this the causative bacteria must be identified more accurately, making it necessary to perform drug-sensitivity tests. Five methods are commonly used to isolate and identify pathogenic bacteria: (i) microscopic examination of smear specimens; (ii) isolation of causative pathogens by culture; (iii) antigen detection; (iv) genetic analysis (diagnostic procedures using DNA and PCR); and (v) determination of antibody titers in serum. 1 There are advantages and disadvantages to each of these methods, and the most suitable method must be chosen for the purpose of the study, because it is senseless to search for causative pathogens blindly. Examination of sputum smears that have persistently colonized in the respiratory tract often cause HAP, making it difficult to The committee for The Japanese Respiratory Society guidelines in management of respiratory infections, Kanda, Chiyoda-ku, Tokyo , Japan. determine whether identified bacteria are truly the causative bacteria or not. In such situations, Gram s stains should be performed to detect predominant bacterial species present with numerous polymorphonuclear cells(pmns). 2,3 Detecting phagocytized bacteria might be considered to support infection. Since the results of smear examinations are greatly influenced by some factors such as examiner s skill, specimen collection methods and patient s condition, the necessity of smear examinations has been controversial. Empirical therapy, however, sometimes leads to unnecessary treatment of non-infectious diseases or to the emergence of resistant bacteria as a result of abuse of antibacterial agents. Moreover, if a variety of bacterial species are isolated from patients with ventilator-associated pneumonia (VAP) and related diseases, causative bacteria can be speculated by determining the predominant bacterial species sometimes phagocytised by numerous PMNs on Gram-stained smears. 3 Before preparing smears, it is very important to carefully examine the characteristics of the sputum specimens. Purulent sputum (sometimes containing blood components) are considered to have originated from pneumonia lesions, and specimens containing large saliva components are unsuitable for microbiological examinations. Miller and Jones classification is a representative system of classification for macroscopic assessment of sputum, while Geckler s classification is a representative method for microscopic assessment of sputum based on gram-staining. Basic Concept for the Management of Community-Acquired Pneumonia in Adults published by the Japanese Res-

2 piratory Society contains the details concerning these classifications. Examination of Gram-stained smear is a simple and rapid method, which is not only useful for assessing the quality of the clinical specimen, but allows identification of the main causative bacteria with a certain degree of confidence. Furthermore, it serves as an important indicator for choosing initial antibacterial agents. On the other hand, specific staining procedures may be required to identify particular bacteria (such as Mycobacterium tuberculosis and Legionella). Fungi may be relatively difficult to isolate/culture from sputum specimens, and pathological approach (Grocott stained and fluorescenceconjugated antibody stained) by trans-bronchial lung biopsy (TBLB) is highly useful for the diagnosis of fungal pneumonia. Cryptococcosis is often complicated by meningitis. When meningitis is suspected, it is important to culture cerebrospinal fluid, and it is also important to perform India ink staining of cerebrospinal fluid specimens. Culture methods for isolation of causative bacteria Since it takes several days to obtain the culture results, they are essentially useless from the standpoint of choosing initial antibacterial agents. However, since drug-resistant opportunistic pathogens have often become a cause of HAP, it is critical to isolate and identify causative bacteria and demonstrate drug sensitivity so as not to complicate treatment. In addition, it is important to verify causative bacteria and to determine their drug-sensitivity for the epidemiological investigation of infectious diseases. At the same time, this information can be used as invaluable data for empirical treatment. For all of these reasons, cultures should be performed at major hospitals when possible. The accuracy of diagnosis is further improved when blood cultures are used in addition to the bacteriological examination of respiratory specimens. Some investigators have reported finding that the blood cultures of 8 20% of patients with HAP were positive and that approximately half of the blood-culturepositive cases with severe pneumonia had infections of other organs. 4 Impressive advances have been made in isolation, culture and identification techniques. It is essential to collect clinical specimens in a timely manner by an appropriate method. On the other hand, some bacterial species require special media, and the detection rate will be higher if the target species are listed on the order forms for bacteriological examination. Detection of pathogen-derived antigens In addition to morphological demonstrations of pathogens, sensitive and rapid procedures are available that allow detection of bacterial antigens and metabolites in clinical specimens. By this immunological means, it is possible to detect bacteria even after the antibacterial treatments. Thus, immunological methods are suitable for searching pathogens in normally aseptic specimens such as cerebrospinal fluid, blood, pleural fluid, and ascitic fluid. It is highly useful when Streptococcus pneumoniae or H. influenzae (b-type) are detected in cerebrospinal fluid and when Streptococcus pneumoniae or Legionella antigen is detected in urine specimens. Several newer immunological techniques are being developed that will allow direct detection of bacterial endotoxins and a variety of enterotoxins, and some new immunological test kits are already commercially available. Molecular (DNA) diagnosis It takes more than one month to grow Mycobacterium tuberculosis in culture, and Legionella and Mycoplasma require special media and nearly a week of culture. Viruses, Rickettsia and Chlamydia require cells and tissues (that serve as a feeder layer) for isolation and identification of the pathogens. There has been great hope that rapid and highly sensitive laboratory procedures would be developed to detect these pathogens. In recent years, tremendous progress has been made in genomic technology, and specific genes or pathogenic genes have been isolated and identified from a number of infectious pathogens, enabling their detection and identification at the molecular levels. Pathogens can now be detected by using DNA probes (by DNA hybridization), and specific DNA probes have been constructed to detect drugresistance regulating genes. DNA diagnosis will become feasible in the future and allow identification of numerous pathogens and pathogen-regulatory factors. The polymerase chain reaction (PCR) method and other amplification procedures have recently been developed. The target DNA fragment (specific DNA) is amplified many times in vitro in a short time (PCR method) with a thermocycler, allowing direct detection of causative bacteria in a scarce specimen. The PCR method has been already used to detect Mycobacterium tuberculosis and several other causative bacteria. However, these procedures are costly, and genomic testing should be done with obvious purpose. Determination of serum antibody titers (serum antibody tests or serology tests) Serum antibody determinations are timeconsuming and almost meaningless in the initial stage of the disease, but they are of great diagnostic significance from the standpoint of retrospective studies. As a matter of fact, a variety of viruses and Legionella are commonly identified by determination of specific antibodies in serum. It is important to confirm the exact diagnosis in terms of the epidemiological aspects of infectious diseases. Furthermore, this effort will provide valuable information for empiric therapy, so clinicians S7

3 S8 should make an effort to confirm the diagnosis as much as possible. DIAGNOSTIC SAMPLING METHODS: NONINVASIVE PROCEDURES AND INVASIVE PROCEDURES, THEIR INDICATIONS AND USES Procedures for sample collection are listed in Table 1. As shown in the table, two major types of procedures (non-invasive and invasive procedures) are commonly employed to collect specimens from the respiratory system. Patients with HAP are often being treated with antibacterial agents or have undergone a variety of treatments, making the disease rather complicated. There is often controversy even in the literature as to whether patients should be subjected to invasive procedures. 2,4 9 If invasive procedures are required, patients may have already been treated with antibacterial agents, or skilful techniques may be required, which often poses problems in clinical practice. Furthermore, there is a report which revealed findings that there was no major difference in mortality according to whether invasive or noninvasive procedures were employed. 6 Since the results of laboratory tests may be influenced (or altered) by the sample collection methods, care should be exercised in the collection of clinical specimens. Endotracheal aspiration is a non-invasive procedure, generally employed in patients who have been intubated. Care should be taken in the diagnosis of HAP, because normal microflora that may not directly reflect the causative bacteria are occasionally detected in sputum samples aspirated from such patients. In such cases, visual observation of predominant bacteria and phagocytosis by Gram s stain is often helpful in making the diagnosis. Quantitative culture tests of aspirated sputum samples allow more accurate identification of causative bacteria, although standard procedure of sample collection and statistically significant bacterial number for the diagnosis Table 1 Procedures for sample collection and their features Non-invasive Expectorated sputum: Collect samples after gargling with tap water Nebulizer-induced sputum: Collect samples after inhalation with a nebulizer Endotracheal aspiration (ETA) Invasive Blood culture Transtracheal aspiration (TTA) Bronchoalveolar lavage (BAL) Protected-specimen brushings (PSB) Transbronchial lung biopsy (TBLB) Percutaneous lung biopsy Open lung biopsy S Kohno et al. have not been established yet. Protected-specimen brushings (PSB) and BAL are available as invasive procedures, and they are generally employed if resistant bacteria, Mycobacterium or fungi are suspected as causative microorganisms. Protectedspecimen brushings and BAL are also often performed in patients who have been intubated. Moreover, if no appropriate secretions are harvested from the respiratory tract, invasive procedures may be needed. Specimens obtained by these procedures can be used for some diagnostic examinations as mentioned above. These procedures are useful diagnostically and are also beneficial in the differential diagnosis, as it is often difficult to differentiate infectious diseases from non-infectious diseases based on the clinical course alone. However, it is difficult to avoid contamination by normal microflora present in the upper respiratory tract, even when BAL is performed. In recent years a new procedure called protected BAL that employs a catheter covered with balloon on its tip has been tried. When quantitative culture tests have been performed by using protected BAL, the sensitivity and specificity of the procedure have been found to be 97% and 92%, respectively, 10 but its use has been rather limited because it is costly. Quantitative culture tests are useful for distinguishing causative bacteria from contaminating bacteria in clinical specimens. However, several issues are still controversial, including patient selection and technical problems in the clinical laboratory. In addition, it is sometimes impossible to identify causative bacteria by determining bacterial number alone in the early stage of pneumonia (at the onset of the disease). Thus, there is still controversy as to whether invasive procedures should be performed. Some investigators have reported sensitivity and specificity value for quantitative culture tests using PSB or BAL in patients with VAP of % and %, respectively. 5,11,12 The presence of bacteria in PSB is considered clinically significant if the colony forming units (CFU) value 10 3 /ml. Similarly, in most hospitals the presence of bacteria in BAL is considered clinically significant if CFU /ml. Table 2 shows an outline of procedures for sample collection and microbiological examinations according to the causative microorganism. MAJOR CAUSATIVE BACTERIA RESPONSIBLE FOR HAP Infection route of HAP may be diverse. For example, HAP may be induced by microaspiration of upper airway secretions, or by aspiration. It may be caused via the air (Legionella, Mycobacterium tuberculosis, fungus and virus) or via blood. Furthermore, iatrogenic transmission can occur by some procedures such as intratracheal intubation and even from the hands of health care workers. By far the most frequent cause of HAP is microaspiration of upper airway secretions. In addition, substantially pathogenic bacteria are usually present in the upper respiratory tract, contributing to the occurrence of HAP. 2 5,9,12 16 Therefore,

4 S9 Table 2 Procedures for sample collection and microbiological examinations according to causative microorganisms 7 Causative microorganism Specimen Examination Staining method Culture Serology Others Aerobic, facultative anaerobic Expectorated sputum, blood, TTA, PSB, BAL, pleural effusion, lung biopsy Gram stain X Anaerobic TTA, pleural effusion, tissue, pus, PSB Gram stain X Legionella Sputum, lung biopsy, pleural effusion, TTA, IFA X IFA PCR, urinary BAL, serum, and urine antigen Nocardia Expectorated sputum, TTA, BAL, tissue, pus Gram stain, modified carbol fuchsin stain Chlamydia Nasopharyngeal swab, serum, lung aspirate, lung biopsy Mycoplasma Expectorated sputum, serum, nasopharyngeal swabs Mycobacterium Expectorated sputum, induced sputum, tissue, gastric juice Fungi Blastomyces, Coccidioides, Histoplasma Expectorated or induced sputum, TTA, BAL, Lung biopsy, Tissue, serum IFA X IFA, CF PCR X X CF, IFA; Cold agglutination method PCR? carbol fuchsin, rhodamine stain X PCR; PPD KOH with phase-contrast GMS stain X CF, ID LA, CF, ID CF, ID Aspergillus Lung biopsy, serum H&E, GMS stain X ID Candida Lung biopsy, serum H&E, GMS stain X ID, LA Cryptococcus Expectorated sputum, serum H&E, GMS stain X LA Zygomycetes Expectorated sputum, tissue H&E, GMS stain X Viruses Nasopharyngeal swabs, lung biopsy, serum IFA X CF, HI, IA neutralisation Pneumocystis Lung biopsy, TTA, BAL, bronchial brushing, induced sputum Toluidine blue, Giemsa, or GMS stain, IFA Note: IFA, immunofluorescent antibody; CF, complement fixation; HI, haemoagglutination inhibition; ID, immunodiffusion; LA, latex agglutination.

5 S10 Table 3 Prevalence of bacteria isolated from sputum collected from patients with hospital-acquired pneumonia (numbers of patients and rates) Number of inpatients S.aureus (26.1%) P.aeruginosa (21.6%) K.pneumoniae (7.6%) E.cloacae 840 (4.0%) X.maltophilia 835 (4.0%) S.marcescens 816 (3.9%) H.influenzae 398 (1.9%) S.pneumoniae 362 (1.7%) A.calcoaceticus 553 (2.7%) E.coli 428 (2.1%) Others (24.4%) Total (86.1%) Reference: the survey on the current status of sensitivity to antibiotics in Japan. S Kohno et al. progress to HAP depends on the species of normal microflora that are present in the nasopharyngeal region. It has been reported in the literature that enteric gram-negative rods (Klebsiella, Proteus, Serratia and E. coli) are detected in the nasopharyngeal region more frequently when hospitalisation is prolonged or the host s condition has deteriorated. Tables 3 and 4 list the major causative pathogens responsible for HAP in Japan. 17,18 The prevalence of Staphylococcus aureus, Pseudomonas aeruginosa, and other gram-negative rods is quite high in Japan. Streptococcus pneumoniae and H. Influenzae are frequently isolated from patients with CAP, and their prevalence is almost as high among patients with HAP in the early period of hospitalisation if they have not been treated with antibacterial agents. However, Streptococcus pneumoniae and H. Influenzae are rarely isolated from patients with HAP who have already been treated with antibacterial agents. Table 5 shows the prevalence of causative microorganisms isolated from patients with HAP. The data in the table are based on a statistical analysis conducted in the US and Europe. Although there are no major differences in the types of causative microorganisms between Japan and the US or Europe, the prevalence of Legionella is lower in Japan. This may be attributable to regional and racial factors, but the low prevalence of Legionella in Japan is probably due to limited diagnostic procedures for Legionella pneumonia. 13 TYPES OF PATHOGENS ACCORDING TO CLINICAL CONDITION In contrast to CAP, which afflicts healthy individuals, HAP frequently occurs in inpatients with underlying disorders, and many of them have experienced use of antibacterial agents and immunosuppressive agents, as well as surgery. The causative bacteria may vary to some extent according to patients background factors and underlying diseases. Treatment must be chosen as appropriate for the causative bacteria, which necessitates a case-by-case approach. 20 Patients with chronic respiratory disease often develop persistent pathogenic bacterial infection in the airway, making it impossible to differentiate causative bacteria from regular colonization on the basis of culture results alone. Gram s stain should be done under such circumstances if patients with chronic respiratory disease develop pneumonia. It is also necessary to identify predominant bacterial species and detect phagocytized bacteria under a light microscope. The prevalence of Pseudomonas aeruginosa, H. influenzae, Klebsiella, and Staphylococcus aureus has been reported to be quite high among these patients. ICU patients and postoperative patients, on the other hand, often develop aspiration pneumonia because they are usually bedridden and on a mechanical ventilator. These patients often develop mixed infections caused by several bacterial species including anaerobic bacteria, frequently complicated by MRSA, Pseudomonas aeruginosa, and Acinetobacter. 2,5,8,15,21,22 In addition, MRSA and non-fermentative gramnegative rods (non-glucose-producing) such as Pseudomonas aeruginosa are frequently isolated from Table 4 Prevalence of bacteria isolated from autopsy lungs with hospital-acquired pneumonia 17 No. of isolated bacteria No. of isolated bacteria P.aeruginosa 67 Trichosporon sp. 3 MRSA 25 Morganella morganii 2 S.aureus 21 Enterobacter aerogenes 2 X.maltophilia 19 Candida tropicalis 2 Enterococcus sp. 16 Nocardia sp. 1 K.pneumoniae 14 Enterococcus faecium 1 Candida albicans 9 Cryptococcus neoformans 1 P.cepacia 7 Acinetobacter sp. 1 Aspergillus fumigatus 4 S.marcescens 1 Enterobacter cloacae 3 Alcaligenes xylosoxidans 1 E.coli 3 Citrobacter freundii 1

6 S11 Table 5 Common causative organisms of hospital-acquired pneumonia 19 Causative organisms Incidence Main source of infection l pneumonia occurring in the early days of hospitalization: Streptococcus pneumoniae 5% 20% Endogenous or other patients Haemophilus influenzae < 5% 15% Airway secretion l pneumonia occurring in the late days of hospitalization: Aerobic gram-negative bacilli 20% 60% Pseudomonas aeruginosa Endogenous Enterobacter spp. Other patients Acinetobacter spp. Environment Klebsiella pneumoniae Tube feeding Serratia Healthcare worker Escherichia coli Medical devices Gram-positive cocci Staphylococcus aureus 20 40% Endogenous, environment, or healthcare workers Pneumonia occurring between the early and late days of hospitalization: Anaerobic cocci 0% 35% Endogenous Legionella 0% 10% Hot water supply, showers, faucets, water tower Mycobacterium tuberculosis < 1% Endogenous, other patients/medical staff Viruses Influenza A, B < 1% Other patients/medical staff Respiratory Syncytial virus < 1% Other patients/medical staff Fungi and protozoa Aspergillus < 1% Air, buildings Candida spp. < 1% Endogenous, other patients/medical staff Pneumocystis carinii < 1% Endogenous, other patients patients who have been treated with antibacterial agents for long periods. 15 Resistant bacteria, including MRSA, b-lactamase-producing species (b-lactamase degrades a broad spectrum cephalosporin antibiotics), and carbapenemase-producing bacteria, have posed clinical problems in recent years. REFERENCES 1 Yamaguchi K. Essential points to remember regarding tests: review of bacteriological tests. Medicina (Tokyo) 1999; 36: Strausbaugh LJ. Nosocomial respiratory infections. In: Mandell GL, Bennett JE, Dolin R (eds) Principles and Practice of Infectious Diseases, 5th edn. Churchill Livingstone, Philadelphia, 2000; Levison ME. Pneumonia, including necrotizing pulmonary infections (lung abscess). In: Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL, Jameson JL (eds) Harrison s Principles of Internal Medicine, 15th Edn. McGraw-Hill, New York, 2001; American Thoracic Society. Hospital-acquired pneumonia in adults: diagnosis, assessment of severity, initial antimicrobial therapy, and preventive strategies. Am. J. Respir Crit Care Med. 1995; 153: Centers for Disease Control. Guidelines for Prevention of Nosocomial Pneumonia. MMWR 1997; 46 (RR-1): Ruiz M, Torres A, Ewig S et al. Noninvasive versus invasive microbial investigation in ventilator associated pneumonia. Am. J. Respir Crit Care Med. 2000; 162: Pennington JE. Respiratory Infections: Diagnosis and Management, 3rd edn. Raven Press, New York, 1994; Fiel S. Guidelines and critical pathways for severe hospital-acquired pneumonia. Chest 2001; 119: 412S 418S. 9 Izumi T, Nagai S, Hamada K. Hospital-acquired pneumonia in adults: diagnosis, assessment of severity, initial antimicrobial therapy, and preventive strategies A consensus report. Ther Res. (Tokyo) 1997; 18: Meduri GU, Beals DH, Maijub AG, Baselski V. Protected bronchoalveolar lavage. Am. Rev. Respir Dis, 1991; 143: Mayhall CG. Hospital Epidemiology and Infection Control. Williams & Wilkins, Philadelphia, PA, 1996; Reese RE, Betts RF. A Practical Approach to Infectious Diseases, 4th edn. Little Brown, Boston, 1996; Mandell LA, Campbell GD Jr. Nosocomial pneumonia guidelines, an international perspective. Chest 1998; 113: 188S 193S. 14 Cook DJ, Walter SD, Cook RJ et al. Incidence of and risk factors for ventilator- associated pneumonia in critically ill patients. Ann. Intern. Med. 1998; 129: Lynch JP. Hospital-acquired pneumonia, Risk factors, microbiology, and treatment. Chest, 2001; 119: 373S 384S. 16 Shinzato T, Saito A. Community-acquired pneumonia and hospital-acquired pneumonia: diagnosis and issues. Sogo Rinsho 1998; 47:

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